Cell lysis device

ABSTRACT

A micromachined cell lysis device with electrodes that are spaced by less than 10 μm from one another. The cells are attracted to the space between the electrodes and then lysed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/954,684, filed Sep. 11, 2001, now U.S. Pat. No. 6,534,295 which is acontinuation of Ser. No. 09/191,268, filed Nov. 12, 1998, now U.S. Pat.No. 6,287,831 which claims the benefit of the U.S. ProvisionalApplication No. 60/065,705, filed on Nov. 14, 1997, which areincorporated herein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

The U.S. Government may have certain rights in this invention pursuantto Grant No. N66001-96-C-8632 awarded by the U.S. Navy.

BACKGROUND

It is known that an electrical field can be used to manipulate cells.Electrical manipulation of cells can be used for separating cells,holding cells, killing micro-organisms, or other operations.

Electrical manipulation of a cell is based on dielectrophoresis. Aneutral particle, such as a microbial cell, will become polarized whensubjected to a non-uniform electric field. Due to the non-uniformity ofthe field, a net force will act on the particle. This force will producemovement of the suspended cell. This phenomenon known asdielectrophoresis the inside of the cell has and holds a differentcharge than the outside of the cell.

Macro sized electroporation systems have been designed for injectinggenes into cells. See “Electroporation and Electrofusion in CellBiology,” E. Newman, A. E. Sauer, C. A. Jordan, ed. Plenum Press, NewYork, 1989. These systems often use electrical fields to make microsizedpores on cell membranes.

Cell lysis typically refers to opening a cell membrane to allow the cellinterior to come out. Cell lysing can be used to obtain intracellularmaterial for further analysis such as DNA identification.

It is known to use the science of micromachining to manipulate cells.See, for example, S. Lee, “A Study of Fabrication and Applications ofMicromachined Cell Manipulating Devices,” Ph.D. Thesis, Seoul NationalUniversity, pp. 77–81, 1996. However, no one has previously reportedusing micromachining to form a device for cell lysis. Usually, thesesystems use cuvets that have a few millimeter range electrode gap.Lysing cells with this kind of size requires a few kilovolts of voltagesource across such a gap.

Prior cell lysing has been reported using pulsed electric fields in amacrosized electroporation system. See, for example, T. Grahl and H.Markl, “Killing of Microorganisms by Pulsed Electric Fields,” Appl.Microbio. Biotechnol., 45, pp. 148–157, 1996. The disadvantages of sucha macrosized device have been described above.

J. Cheng, et al, “Preparation and Hybridization analysis of DNA/RNA fromE. Coli on Microfabriacted Bioelectronic Chips” has suggested electroniccell lysis on a chip. However, this system still required hundreds ofvolts for lysing the cell.

SUMMARY

The present disclosure describes a new micromachined cell lysis device.A microsized cell lysis device as disclosed reduces the size of theentire system including the power source, since the electrode gap couldbe reduced to a few μm or smaller. This micro-sized cell lysis device iscapable of operating on a small number of cells due to its small size.

A special way of using the electric field that can greatly simplify thepurification steps is described. This can be used to prepare biosamples.In addition, the small size allows a reduction in voltage required forlysing. The voltage can be reduced to practical levels, e.g., less than50 volts, since the electrode gap is on the order of microns.

A new structure is also described for cell lysis.

BRIEF DESCRIPTION OF THE DRAWING

These and other advantages will now be described in detail with respectto the accompanying drawings, wherein:

FIG. 1A shows a schematic view of the overall cell lysis device;

FIG. 1B shows a top view of the cell lysis electrode;

FIGS. 2A–2D show the fabrication steps of the cell lysis device;

FIG. 3 shows a photograph of a fabricated device;

FIG. 4 shows schematically the power system used for cell lysis;

FIG. 5 shows a plot of a waveform for cell lysis;

FIG. 6 shows drawings of yeast cells before and after lysing;

FIG. 7 shows a plot of lysis vs voltage.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The basic lysis device is shown in plan view in FIGS. 1A and 1B. Thedevice is made according to the fabrication steps explained below withreference to FIGS. 2A–2D.

The micromachining operates to form features on a silicon substrate.

First an insulator is formed on the silicon substrate, by oxidizing thesilicon substrate 200 to form a thermally-grown 5000 Å silicon oxidelayer 201 as shown in FIG. 2A. Chromium/gold (Cr/Au) is thermallyevaporated and patterned to form electrodes 202 on the oxidized surface.The electrodes are formed with a number of pointed portions facing oneanother, in the general shape shown in FIG. 1B.

A 4 μm thick layer of PARYLENE ™ type parax-ylylene layer is depositedand patterned to form Parylene barriers 210 as shown in FIG. 2C. Thesebarriers have side surfaces that hold the cell in a proper place, andform blocks between each pair of electrode surfaces.

FIG. 2D shows bonding the thus-made assembly to a glass substrate whichhas an inlet 220, an outlet 222, and a channel 230 between the inlet andoutlet. The channel is 30 μm high, made by timed wet etching.

The preferred device is designed for yeast cells. The distance betweenelectrodes is hence around 5 μm. More generally, the distance can rangebetween about 0.8 μm and 100 μm (0.1 mm), more preferably on the orderof e.g. 1–9.9 μm.

The final assembled device is shown in FIG. 1A. A number of cells areshown, such as cell 102. Cells are attracted by the dielectrophoreticforce using an AC voltage. The cells are then lysed, using pulsedelectric fields. The AC voltage depends on the conductivity andpermitivity of the cell suspensions and the sizes of the cells. Thecells are held between two electrodes 110, 112 and between the PARYLENE™ type para-xylylene barriers 120, 122 for the lysing.

Any arrangement of pairs of electrodes, such as interdigitated orparallel, can be used for the cell lysing. Preferably, the edges of theelectrodes are made sharp as shown in order to concentrate the fieldbetter on the cells. The nearest distance 114 between the two electrodesis preferably equal to the mean diameter of a cell plus the standarddeviation of the cells in order to obtain the most effective lysing.

FIG. 1A shows a drawing of the electrode without the PARYLENE ™ typepara-xylylene barriers present showing interdigitated electrodes.Distance 114 is defined as the distance between the sharp ends of theelectrodes.

FIG. 3 shows a drawing of the device from the top, showing all thearrangements of the various structures.

An important feature includes how the device is operated. A power systemfor the cell lysis is formed as shown in FIG. 4. Control is selected bya switch 400 which selects between manual mode or automatic mode. In themanual mode, the pulse is applied by a push-button switch 402. In theautomatic mode, pulses are supplied at every defined interval. Pulsewidth control is provided by a multivibrator 410, typically a TTL-typemultivibrator, part 74LS123. The switch 400 can be a single-pull,double-throw type relay.

A multipurpose function generator 420 provides the electric fields whichattracts the cells. The electric field is preferably a sinusoidal wave.A power MOSFET 422 provides the output to the cell lysis device 100.

A typical waveform is shown in FIG. 5, which shows a sample plot of thewaveform for cell lysis. The waveform includes two parts—the attractionphase 500, and the lysing phase 502.

The attraction phase uses a 6 volt AC, 2 MHZ sample. This attracts thecells to the lysing locations. A sinusoidal wave is preferably used toattract the cell to the location. After a short delay, lysing pulse, a100 μs, 20 volt pulse, is applied.

FIGS. 6A and 6B show the yeast cells before and after applying thepulsed voltage. FIG. 6A shows attraction of the yeast cells to theelectrode when the 2 MHZ 6V AC voltage in FIG. 5 is applied. FIG. 6Bshows the result of lysing. After lysing the cells, the inside andoutside of the cells are electrically connected, and they will no longerattract to the electrodes by the AC voltage.

FIG. 7 shows some representative lysing rates with different electricfields and pulse durations. The rate is increased with increased voltageand duration. Excessive pulse voltage and duration form electrolysiseffects. The optimum value for yeast cell lysing is believed to occur at100 μs and 20V. However, any voltage less than 50 volts is preferred andwithin the preferred embodiment.

Although only a few embodiments have been described in detail above,other embodiments are contemplated by the inventor and are intended tobe encompassed within the following claims. In addition, othermodifications are contemplated and are also intended to be covered. Forexample, other shapes and sizes of electrodes could be used. There couldalso be more than two electrodes. While the pointed electrodes arepreferred, flat shaped electrodes can also be used.

1. A cell lysis device, comprising: an insulator on a substrate; a pair of conductors, formed on the insulator, each said conductor being formed with substantially sharp pointed electrodes with pointed portions on one of said conductors that face corresponding pointed portions on the other of said conductors, and which pointed portions are separated from one another by a specified distance between 0.8 μm and 100 μm; a plurality of barriers, wherein each one of said plurality of barriers is placed on top of one of said conductors so that it does not cover said pointed portions; and an electrical connection to said pair of conductors.
 2. A device as in claim 1, wherein said insulator is an oxide layer.
 3. A device as in claim 2, wherein the substrate comprises silicon.
 4. A device as in claim 1, wherein said barriers are formed of para-xylylene.
 5. A device as in claim 1, wherein said barriers are substantially rectangular in cross-section, forming walls adapted for containing cells.
 6. A device as in claim 1, wherein said conductors are formed of chromium/gold.
 7. The device as in claim 1, further comprising a power supply which forms a dielectrophoretic force to attract cells.
 8. The device as in claim 7, wherein the power supply produces pulsed electric fields after producing the dielectrophoretic force. 